The intestinal tract is exposed to numerous commensal bacteria, and is an important entry portal for clinically relevant bacterial pathogens. Toll-like receptors (TLRs) are a family of receptors that recognize bacterial products and, once activated, can initiate innate immune defenses in the host. TLR9 recognizes bacterial DNA (bDNA) and its synthetic derivatives carrying an unmethylated CpG motif, which is absent from mammalian DNA. Functional TLR9 is expressed by various immune and inflammatory cells (macrophages, B cells), and, as shown in preliminary studies, also in intestinal epithelial cells. Administration of bDNA to mice can be immunostimulatory (e.g., adjuvant functions in immunizations), but can also downregulate inflammatory responses, as we showed in murine colitis models. To explain these different effects ofbDNA, when administered exogenously or after release from pathogenic and commensal intestinal bacteria, this Research Unit will focus on the physiologic functions of the receptor for bDNA, TLR9, in the intestine. Our studies will test the overall hypothesis that TLR9 exerts anti-inflammatory functions in the intestine in vivo. The studies have the following Specific Aims: Aim 1. To define the signaling pathways activated by bDNA in intestinal epithelial cells. We will define the polarity and subcellular localization of TLR9 in polarized human intestinal epithelial cells in vitro and in vivo, and determine the signaling pathways and cellular functions induced by TLR9 activation in these cells. Aim 2. To determine the functions of TLR9 in regulating colonic inflammation. These studies will use TLR9 deficient mice to define the physiologic functions of TLR9 in different murine colitis models, and in mediating the beneficial effects ofprobiotic bacterial preparations in colitis. Aim 3. To define the importance of indoleamine 2,3-dioxygenase (IDO) in mediating the anti-inflammatory functions of bDNA in colitis. Based on preliminary data that inhibition of the tryptophan-catabolizing enzyme, IDO, reverses the anti-inflammatory effects of bDNA, the studies will use IDO deficient mice and pharmacological and histological approaches to begin to address mechanisms by which bDNA and TLR9 modulate intestinal inflammation. Together, these studies will yield new insights into the physiologic functions of bacterial DNA and TLR9 in the intestine, which will form a basis for the design of new therapeutic strategies that exploit the underlying biological principles.